Transformation of Hematopoietic Cells and Activation of JAK2-V617F by IL-27R, a Component of a Heterodimeric Type I Cytokine Receptor
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Transformation of hematopoietic cells and activation of JAK2-V617F by IL-27R, a component of a heterodimeric type I cytokine receptor Anuradha Pradhan, Que T. Lambert, and Gary W. Reuther* Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612 Edited by Stephen P. Goff, Columbia University College of Physicians and Surgeons, New York, NY, and approved October 5, 2007 (received for review March 14, 2007) From a patient with acute myeloid leukemia (AML), we have shown to be leukemogenic in mice (9), further validating our identified IL-27Ra (also known as TCCR and WSX1) as a gene whose approach to identify genes that contribute to leukemia formation. expression can induce the transformation of hematopoietic cells. In this study, we have used a functional genetic screen to IL-27Ra (IL-27R) is a type I cytokine receptor that functions as the identify genes that contribute to myeloid cell transformation and ligand binding component of the receptor for IL-27 and functions perhaps AML formation. Here, we describe our identification of with the glycoprotein 130 (gp130) coreceptor to induce signal the ligand binding component of the receptor for IL-27 (IL- transduction in response to IL-27. We show that IL-27R is expressed 27R), as a unique transforming gene product. We show that on the cell surface of the leukemic cells of AML patients. 32D IL-27R is expressed on the cell surface of leukemic cells of AML myeloid cells transformed by IL-27R contain elevated levels of patients and demonstrate that IL-27R can transform hemato- activated forms of various signaling proteins, including JAK1, JAK2, poietic cells in a JAK-dependent manner. We also show that STAT1, STAT3, STAT5, and ERK1/2. Inhibition of JAK family proteins IL-27R activates JAK2-V617F. Previously, only homodimeric induces cell cycle arrest and apoptosis in these cells, suggesting the type I cytokine receptors have been reported to activate this transforming properties of IL-27R depend on the activity of JAK important JAK2 mutant. Our data indicate that a single com- family members. IL-27R also transforms BaF3 cells to cytokine ponent of a heterodimeric cytokine receptor has the ability to independence. Because BaF3 cells lack expression of gp130, this aberrantly activate signaling pathways in hematopoietic cells. finding suggests that IL-27R-mediated transformation of hemato- Thus, it is possible that components of heterodimeric cytokine poietic cells is gp130-independent. Finally, we show that IL-27R can receptors may play a role in myeloid diseases either through functionally replace a homodimeric type I cytokine receptor in the aberrant expression or activating mutations. activation of JAK2-V617F, a critical JAK2 mutation in various myeloproliferative disorders (MPDs). Our data demonstrate that Results IL-27R possesses hematopoietic cell-transforming properties and Functional Genetic Screens to Identify Myeloid Cell Oncogenes Un- suggest that, analogous to homodimeric type I cytokine receptors, cover Unique Transforming Properties of IL-27R. To identify onco- single-chain components of heterodimeric receptors can also en- genes involved in AML, or perhaps other myeloid disorders, we hance the activation of JAK2-V617F. Therefore, such receptors may developed functional genetic screens in myeloid cells. We used play unappreciated roles in MPDs. 32D myeloid progenitor cells, which depend on IL-3 for growth and viability (10). When 32D cells are cultured in the absence of acute myeloid leukemia ͉ myeloproliferative disease ͉ oncogene ͉ IL-3, they undergo cell cycle arrest and apoptosis (11). These signaling cells have served as a model cell system for studying the transforming properties of various leukemia-associated onco- cute myeloid leukemia (AML) is a disease of the myeloid genes, such as Bcr/Abl and Flt3, among others (12, 13). For our Acompartment of the hematopoietic system characterized by approach, we used cDNA representing the genes expressed in an accumulation of undifferentiated blast cells in the peripheral the leukemic cells of patients with AML. This cDNA was cloned blood and bone marrow. AML is caused by multiple genetic and into a retroviral vector (pEYK3.1; a gift of George Daley, epigenetic changes that result in stimulation of mitogenic signals Harvard Medical School, Children’s Hospital, Boston) designed and deregulation of apoptosis and differentiation. It has been for efficient delivery and proviral recovery (14). We generated proposed that mutations in two different classes of oncogenes retrovirus containing cDNA libraries from AML patients and are required to induce AML. Mutations in class I oncogenes used this virus to infect 32D cells that exogenously express B-cell (e.g., Ras, Flt3) result in stimulation of proliferative and cell CLL/lymphoma 2 (Bcl2). survival signals, whereas mutations in class II oncogenes (e.g., We used 32D/Bcl2 cells for multiple reasons. First, removal of AML1-Eto, PML-RAR␣) lead to inhibition of differentiation and IL-3 leads to a very rapid apoptotic cell death of 32D cells, and subsequent cell death by apoptosis (1–3). Although many mu- our screens of parental 32D cells resulted in no cytokine- tations are recurrently found in AML patients, it is believed that independent isolates. Second, oncogenic expression in 32D cells additional mutations in AML exist and have yet to be identified needs to elicit an antiapoptotic and a mitogenic signal to (1, 2, 4–6). Even with a large knowledge base about the causative transform these cells to cytokine independence. Third, it is genetics of AML, a more complete understanding of the mo- lecular players is needed to identify targets for future therapeutic treatment for this disease. Author contributions: A.P. and G.W.R. designed research; A.P., Q.T.L., and G.W.R. per- We have previously performed functional genetic screens to formed research; A.P., Q.T.L., and G.W.R. analyzed data; and G.W.R. wrote the paper. identify oncogenes in AML by using an efficient retroviral The authors declare no conflict of interest. delivery, expression, and cDNA recovery system (7, 8). Using This article is a PNAS Direct Submission. this approach, we identified an activating deletion mutation in *To whom correspondence should be addressed. E-mail: gary.reuther@moffitt.org. the TrkA tyrosine kinase in a patient with AML (7). This This article contains supporting information online at www.pnas.org/cgi/content/full/ discovery provided evidence that TrkA may play a role in 0702388104/DC1. leukemogenesis. The deletion mutation we identified has been © 2007 by The National Academy of Sciences of the USA 18502–18507 ͉ PNAS ͉ November 20, 2007 ͉ vol. 104 ͉ no. 47 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702388104 Downloaded by guest on September 28, 2021 Fig. 1. IL-27R transforms 32D cells to IL-3 independence. (A) 32D cells stably expressing IL-27R (F) or a control vector (■) were cultured in the absence of IL-3 starting on day 0. The total number of viable cells was determined at each time point by trypan blue exclusion. The dotted line represents the total number of viable cells going below the limit of detection of a hemacytometer and to zero. (B) Cell lysates of 32D cells stably expressing a control vector (lane 1) or IL-27R (lane 2) were immunoblotted with anti-IL-27R antibodies. (C) Antibodies that recognize the indicated proteins were used to immunoblot lysates from control 32D cells expressing an empty vector (lanes 1 and 3) or IL-27R (lanes 2 and 4) after1hofgrowth factor starvation. (D) Bone marrow mononuclear cells from normal and AML patients were analyzed by flow cytometry using an Alexa Fluor-647-conjugated anti-IL-27R antibody. The percentage of IL-27R-positive cells of each patient sample (represented by F) is plotted. believed that transformation of myeloid cells in AML is caused STAT1, STAT3, and STAT5 are frequently activated by leuke- by mutations that activate cooperating signaling pathways (1–3). mogenic oncogenes and are activated in AML (22, 23). JAK Culturing 32D cells without IL-3 resulted in cell death of the proteins, in particular JAK2, are mutationally activated in entire culture in a little over 2 days. However, expression of Bcl2 myeloproliferative disorders (MPDs) (24–28) and a small frac- led to enhanced cell survival in the absence of IL-3 without tion of AML (29, 30). Therefore, we analyzed the activation state inducing cell growth (data not shown), which is in agreement of these proteins in 32D cells transformed by IL-27R and with previous studies (15, 16). Two days after infection, 32D/Bcl2 determined that these cells have increased phosphorylated cells were plated in medium lacking IL-3 to identify IL-3- STAT1, STAT5, JAK1, and JAK2 compared with vector control independent transformants. Using a cDNA library from an cells (Fig. 1C). STAT3 is also activated in 32D cells transformed AML patient who exhibited AML of FAB subtype M5b, a by IL-27R (see Fig. 2E). Because transformation of 32D cells monocytic leukemia with 93% blast cells and normal cytogenet- involves mitogenic signaling and inhibition of apoptosis, we also ics, we isolated a cDNA representing a WT IL-27Ra (TCCR, determined that ERK1/2 is activated in these cells (Fig. 1C). WSX-1) (17) gene from multiple independent isolates of IL-3- These data indicate that IL-27R-mediated transformation of independent cells. For simplicity, we will refer to this gene and 32D cells is associated with activation of JAK/STAT and ERK protein as IL-27R. pathways. In addition to STATs, ERKs are commonly activated in AML cells (31). Thus, transformation by IL-27R results in Transformation of 32D cells by IL-27R. Expression of IL-27R in activation of pathways that are frequently activated in AML. CELL BIOLOGY 32D/Bcl2 cells is sufficient to transform these cells to IL-3 Activation of transforming signals by IL-27R is not associated independence (data not shown).